Technical Abstract: Systemic uptake bioassays using excised cotton leaves confirmed resistance to imidacloprid in a Guatemalan population of the tobacco whitefly Bemisia tabaci Gennadius. Polyacrylamide gel electrophoresis of naphthyl esterases identified the insects as B-types. Upon collection from the field, resistance was determined to be 58-fold relative to a susceptible strain originating in the Imperial Valley of California. Resistance levels increased to 126-fold in this population during its continuous exposure to systemically treated cotton. In biochemical investigations, there was no detectable NADPH-dependent mixed function oxidase metabolism of 14C-imidacloprid at any time during the selection process. In contrast, microsomal preparations from housefly abdomens readily produced significant amounts of the mono-hydroxy and olefin derivatives of the parent compound. Detoxification of imidacloprid by housefly MFOs may account for reports of lower toxicity of the insecticide towards this insect compared with whiteflies, despite similar binding properties between imidacloprid and the nicotinic acetylcholine receptors in both species.